Allele-specific amplification of nucleic acids allows for simultaneous amplification and analysis of the target sequence. Allele-specific amplification is commonly used when the target nucleic acid has, or is suspected of having, one or more subpopulations with a variation (polymorphism) in its sequence. DNA polymorphisms are used in DNA profile analysis (forensics, paternity testing, tissue typing for organ transplants), genetic mapping, distinguishing between pathogenic strains of microorganisms, as well as detection of rare mutations, such as those occurring in cancer cells in the background of cells with normal DNA.
In a successful allele-specific amplification, the desired variant of the target nucleic acid is amplified, while the other variants are not, at least not to a detectable level. A typical allele-specific amplification assay involves a polymerase chain reaction (PCR) with at least one allele-specific primer designed such that primer extension occurs only when the primer forms a hybrid with the desired variant of the target sequence. When the primer hybridizes to an undesired variant of the target sequence, primer extension is inhibited.
Certain improvements for allele-specific amplification of nucleic acid targets have been proposed. Further improvements in the effectiveness and sensitivity of allele-specific amplifications for many clinically-relevant nucleic acid targets of nucleic acid targets are desirable, and the present disclosure provides related benefits.